Polyketides represent a large family of diverse compounds synthesized from 2 carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms including fungi and mycelial bacteria, in particular the actinomycetes. An appreciation for the wide variety of polyketide structures and for their biological activities may be gained upon review of the extensive art, for example, published International Patent Specification WO 95/08548; U.S. Pat. Nos. 5,672,491 and 6,303,342; Fu et al., 1994, Biochemistry, 33:9321-26; McDaniel et al., 1993, Science, 262:1546-50; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34:881-88.
Polyketides are synthesized in nature by polyketide synthases (“PKS”). These synthase enzymes are complexes of multiple enzyme activities. Two major types of PKS are known and differ in their mode of synthesis. These are commonly referred to as Type I or “modular” and Type II “iterative.” The Type I or modular PKSs, as commonly found in bacteria but not in fungi, comprise a set of separate catalytic active sites; the portion of the protein that encompasses each active site region is termed a “domain”, and a set thereof is termed a “module”. One module exists for each cycle of carbon chain elongation and modification. FIG. 9 of aforementioned WO95/08548 depicts a typical Type I PKS, in this case 6-deoxyerythronolide B synthase (“DEBS”) which is involved in the production of erythromycin. Six separate modules, each catalyzing a round of condensation and modification of a 3-carbon unit, are present in DEBS. The number and type of catalytic domains that are present in each module varies based on the needed chemistry, and the total of 6 modules is provided on 3 separate polypeptides (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules per each polypeptide). Each of the DEBS polypeptides is encoded by a separate open reading frame (gene), see Caffrey et al., 1992, FEBS Letters, 304:205. DEBS provides a representative example of a modular Type I PKS. In DEBS, modules 1 and 2 reside on DEBS-1, modules 3 and 4 on DEBS-2, and modules 5 and 6 on DEBS-3, wherein module 1 is defined as the first module to act on the growing polyketide backbone, and module 6 the last.
The minimal PKS module is typified by module 3 of DEBS which contains a ketosynthase (“KS”) domain, an acyltransferase (“AT”) domain, and an acyl carrier protein (“ACP”) domain. These three enzyme activities are sufficient to activate a 2, 3, or more -carbon extender unit and attach it to the growing polyketide molecule. Additional domains that may be included in a module relate to reactions other than the actual condensation, and include domains for a ketoreductase activity (“KR”), a dehydratase activity (“DH”), and an enoylreductase activity (“ER”) and a methyltransferases activity. With respect to DEBS-1, the first module thereof also contains additional AT and ACP domains because that module catalyzes the initial condensation, and so begins with a “loading di domain” (sometimes referred to as a loading module) that contains an AT and ACP, that bind the starter unit. The “finishing” of the 6-deoxyerythronolide molecule is regulated by a thioesterase activity (“TE”) in module 6 that catalyzes cyclization of the macrolide ring during release of the product of the PKS.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or “scaffold”-encoding regions. These scaffold regions encode amino acid sequences that space the enzymatic activities (domains) at the appropriate distances and assure the correct order of modules in the PKS. Thus, these linker regions collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the level of encoding DNA) from other PKS by various available methodologies. Thus, there is considerable flexibility in the design of a new PKS to produce a novel polyketide. An additional level of structural complexity in the resultant polyketides may be introduced by subsequent P450 oxidation, methylation, glycosylation or other enzymes that catalyze post-PKS reactions.
Geldanamycin is a polyketide produced by a modular PKS and was the first of four benzoquinone ansamycins isolated from microorganisms (see FIG. 1) to have been evaluated extensively as an antitumor drug. Although originally discovered by screening microbial extracts for antibacterial and antiviral activity [DeBoer, et al. (1970), Sasaki, et al. (1970); full citations of all references cited herein by the author and year of publication are provided below], geldanamycin was later found to be cytotoxic to tumor cells in vitro [Sasaki et al. (1979)] and to reverse the morphology of cells transformed by the Rous sarcoma virus to the normal state [Uehara et al. (1986)]. Subsequent discoveries of the herbimycins [Omura et al. (1979)], macbecins [Muroi et al. (1980)] and TAN 420A [Shibata et al. (1986)] expanded this class of antitumor natural products. Ansamycins like the ansamitocins are usually included in this class of microbial products. Reblastatin, isolated from the geldanamycin producer, was recently found to have interesting biological activities [Takatsu et al. (2000), Stead et al. (2001)].
Geldanamycin's nanomolar potency and apparent specificity for aberrant protein kinase dependent tumor cells, as well as the discovery that its primary target in mammalian cells is the ubiquitous Hsp90 protein chaperone, has stimulated interest in its development as an antitumor drug [Neckers et al. (2002); Blagosklonny, 2002]. Severe hepatotoxicity [Supko et al. (1995)] led to its withdrawal from Phase I clinical trials in 1995. Nonetheless, during the 1990's considerable information was obtained about the structure-activity relationships (SAR) of geldanamycin, herbimycin and reblastatin [Neckers et al. (2002), Schnur et al. (1995)]. In late 1999, 17-allylamino-17-desmethoxygeldanamycin entered Phase I clinical trials [Egorin et al. (2001), Wilson et al. (2001), Erlichman et al. (2001)] sponsored by the National Cancer Institute in the US and the Cancer Research Campaign in the UK because this analog had exhibited good in vivo activity [Wilson, et al. (2001), Erlichman, et al. (2001)], better pharmacokinetics and lower toxicity than geldanamycin [Egorin et al. (2001)] during preclinical development. The maximum tolerated dose is 40 mg/m2 [Wilson et al. (2001)], and micromolar serum concentrations are achieved without overt toxicity. Efficacy in inhibiting signal transduction pathways has been demonstrated in peripheral blood lymphocytes.
There is therefore a need for recombinant nucleic acids, host cells, and methods of expressing those nucleic acids in host cells to produce geldanamycin at a commercially useful scale and to make geldanamycin analogs. These and other needs are met by the materials and methods provided by the present invention.
The following articles provide additional background information relating to the invention and are incorporated herein by reference. DeBoer et al. “Geldanamycin, a new antibiotic” J Antibiot (Tokyo) (1970) 23:442-7. Sasaki et al. “Geldanamycin. I. Structure assignment” J Am Chem Soc (1970) 92:7591-3. Blagosklonny, 2002, “Hsp-90-associated oncoproteins: multiple targets of geldanamycin and its analogs” Leukemia 16:455-62. Sasaki et al. “Growth inhibition of virus transformed cells in vitro and antitumor activity in vivo of geldanamycin and its derivatives” J Antibiot (Tokyo) (1979) 32:849-51. Uehara et al. “Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus” Mol Cell Biol (1986) 6:2198-206. Omura et al. “Herbimycin, a new antibiotic produced by a strain of Streptomyces.” J Antibiot (Tokyo) (1979) 32:255-61. Iwai et al. “Herbimycin B, a new benzoquinonoid ansamycin with anti-TMV and herbicidal activities” J. Antibiot (Tokyo) (1980) 33:1114-9. Muroi et al. “Macbecins I and II, new antitumor antibiotics. II. Isolation and characterization” J Antibiot (Tokyo) (1980) 33:205-12. Shibata et al. “The structure and cytocidal activity of herbimycin C.” J Antibiot (Tokyo) (1986) 39:1630-3. Takatsu et al. “Eblastatin, a novel benzenoid ansamycin-type cell cycle inhibitor.” J. Antibiot. (2000) 53:1310-1312. Stead et al. “Discovery of novel ansamycins possessing potent inhibitory activity in a cell-based oncostatin M signalling assay. J. Antibiot (Tokyo) 53:657-663. Neckers, L. “Hsp90 inhibitors as novel cancer chemotherapy agents.” Tr. Molec. Med. (2002) 8:S55-S61. Supko et al. “Preclinical pharmacologic evaluation of geldanamycin as an antitumor agent.” Cancer Chemother Pharmacol (1995) 36:305-15. Schnur, et al. “erbB-2 Oncogene inhibition by geldanamycin derivatives: synthesis, mechanism of action, and structure-activity relationships.” J. Med. Chem. (1995) 38:3813-20. Egorinet et al. “Plasma pharmacokinetics and tissue distribution of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) in CD2F1 mice.” Cancer Chemother Pharmacol (2001) 47:291-302. Wilson et al. “Phase I pharmacologic study of 17-AAG in adult patients with advanced solid tumors.” Amer Soc of Clin Oncol, (2001) Abstract 325; Erlichman et al. “A phase I trial of 17-AAG in patients with advanced cancer.” Proceedings of the AACR (2001) Abstract. Guo J, Frost J W. “Biosynthesis of 1-deoxy-1-imino-D-erythrose 4-phosphate: (2002). A defining metabolite in the aminoshikimate pathway.” (2002) J Am Chem Soc. 124, 528-9. Yu et al. (2002). “The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum.” Proc Natl Acad Sci USA. 99, 7968-73. August et al. (1998). “Biosynthesis of the ansamycin antibiotic rifamycin: deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699.” Chem Biol 5, 69-79; Leistner E (1999). “Biosynthesis of ansatrienin (mycotrienin) and naphthomycin. Identification and analysis of two separate biosynthetic gene clusters in Streptomyces collinus Tu1892.” Eur J Biochem 261, 98-107; DeBoer C, Dietz A. (1976). “The description and antibiotic production of Streptomyces hygoscopicus var. geldanus.” J Antibiot 29, 1182-8. Kunkel, T. A. Proc Natl Acad Sci USA (1985) 82:448. Geisselsoder et al. BioTechniques (1987) 5:786. Zoller and Smith, Methods in Enzymology (1983) 100:468. Dalbie-McFarland et al. Proc Natl Acad Sci USA (1982) 79:6409.